Autophagy-related genes genetically interact with Pmk1 MAPK signaling in fission yeast

Apart from the highly conserved role in the cellular degradation process, autophagy also appears to play a key role in cellular proliferation. Here, we describe the genetic interaction of autophagy-related genes and Pmk1 MAPK signaling in fission yeast. atg1 deletion cells (Δ atg1 ) exhibit the vic (viable in the presence of immunosuppressant and Cl - ) phenotype, indicative of Pmk1 signaling inhibition. Moreover, the Δ atg1 Δ pmk1 double mutant resembles the single Δ pmk1 mutant, suggesting that Atg1 functions in the Pmk1 pathway. In addition, the growth defect induced by overexpression of Pck2, an upstream activator of Pmk1 MAPK was alleviated by the deletion of atg1 + . Finally, the deletion of autophagy-related genes recapitulates Pmk1 MAPK signaling inhibition. Our data suggest a novel role for autophagy in MAPK signaling regulation.


Figure 1. Deletion of autophagy-related genes phenotypically recapitulates Pmk1 MAPK signaling inhibition
A: Strains grown in EMM with leucine (EMML) were serially diluted and spotted onto EMML plates with or without FK506 and 0.09 M MgCl 2 , then incubated at 27˚C for 4 days. B: Strains grown in EMM with thiamine were serially diluted and spotted onto EMM plates with or without thiamine, then incubated at 27˚C for 7 days. C: WT, pmk1 deletion cells (Δpmk1), and cells deleted for autophagy-related genes were grown in YPD and serially diluted and spotted onto YPD plates with or without FK506 and 0.09 M MgCl 2 , then incubated at 27˚C for 3 days.

Description
Autophagy is an evolutionarily highly conserved mechanism to maintain cellular homeostasis via waste clearance using the lysosomal machinery (Mukaiyama et al. 2010;Mizushima and Komatsu 2011). Although starvation potently induces autophagy, the basal level of autophagy is maintained even in normal growth conditions, thereby controlling development, cellular metabolism, and proliferation in all eucaryote cells (Wang and Levine 2010;Musiwaro et al. 2013;Kim and Lee 2014;Birkenmeier et al. 2016;Allen and Baehrecke 2020). However, how autophagy organizes cellular signaling networks to exert these functions is not fully understood.
Here, we identified atg1 + , a critical component of the autophagy machinery, as a gene that functions in the Pmk1 MAPK signaling pathway in fission yeast via a molecular genetic screening utilizing the vic (viable in the presence of immunosuppressant and chloride ion) phenotype, which recapitulates Pmk1 MAPK signaling inhibition (Methods). The rationale of the vic mutant screening is based on the antagonistic relationship between Pmk1 MAPK and calcineurin, a highly conserved serine/threonine protein phosphatase, in the Cl − homeostasis in fission yeast. Knockout of the S. pombe calcineurin gene ppb1 + or inhibition of calcineurin activity by the immunosuppressant FK506, a specific inhibitor of calcineurin, results in hypersensitivity to Cl − (Sugiura et al. 1998). This phenotype associated with ppb1 deletion was reversed by the inhibition or gene knockout of the components of the Pmk1 MAPK signaling pathways, including Pmk1 MAPK, Pek1 MAPKK, Mkh1 MAPKKK, and Pck2 Protein kinase C (Sugiura et al. 1998(Sugiura et al. , 1999Ma et al. 2006). Our previous genetic screening to isolate vic mutants identified upstream activating regulators of MAPK signaling such as geranylgeranyl transferase (Doi et al. 2015) and farnesyl transferase (Ma et al. 2006).
The growth of the wild-type (WT) cells was significantly inhibited in the presence of the calcineurin inhibitor FK506 and 0.09 M MgCl 2 , whereas knockout of the pmk1 + gene makes cells grow much faster in the same condition ( Figure 1A, Δpmk1). Δatg1 cells grew faster than the WT cells in the presence of FK506 and 0.09 M MgCl 2 , although the growth of Δatg1 cells was slower than that of Δpmk1 ( Figure 1A, Δatg1). The WT, Δpmk1 and Δatg1 cells exhibited essentially the same pattern of sensitivity and resistance to FK506 and Cl − on EMM, YPD and YES plates, although the sensitivity of the WT cells and Δatg1 cells was more enhanced on YES than the other plates. Genetic interaction between Δatg1 and Δpmk1 was further examined by constructing Δatg1Δpmk1 double mutant cells. The degree of the vic phenotype in Δatg1Δpmk1 double mutants and Δpmk1 cells was almost equivalent. These results are consistent with Atg1 working upstream of the Pmk1 pathway. The difference in growth of these strains is almost indiscernible in the absence of FK506 and MgCl 2 ( Figure 1A).
To further explore the functional relationship between Atg1 and Pmk1 MAPK signaling, we utilized the cell growth inhibition associated with Pck2 overexpression. Pck2 overexpression in the WT cells leads to Pmk1 MAPK hyperactivation and cytotoxicity, which can be suppressed by the inhibition or knockout of the components of the Pmk1 MAPK pathway ( Figure  1B) (Ma et al. 2006). This phenotypic evaluation also led to the identification of an SH3 adaptor protein Skb5 as a negative regulator of Pck2/Pmk1 signaling (Kanda et al. 2016). As shown in Figure 1B, atg1 deletion significantly suppressed the toxicity induced by Pck2 overexpression, although the impact of atg1 deletion on the suppression of the toxicity of Pck2 overexpression was smaller than that achieved by pmk1 deletion. Thus, atg1 deletion is likely to ameliorate Pck2-mediated Pmk1 MAPK hyperactivation.
Next, we confirm if the loss-of-function mutants of other autophagy-related genes also display the vic phenotype. As expected, a series of the deletion mutants of autophagy-related genes, except for atg15 + , grew better than the wt strain in the media containing FK506 plus 0.09 M MgCl 2 ( Figure 1C). The degree of the vic phenotype of these atg mutants was similar to that of Δatg1 cells. These results suggest that the autophagy system as a whole may be involved in the Pmk1 MAPK signaling regulation.
In summary, our genetic screen revealed a functional interaction between autophagy-related genes and Pmk1 MAPK signaling in fission yeast. Several studies report functional crosstalk between MAPK signaling and autophagy, including the role of MAPK ERK in the maturation of autophagosomes (Corcelle et al. 2006), as well as the role of autophagosome as a scaffold to facilitate spatial coordination of RAF/MEK/ERK phosphorylation (Martinez-Lopez et al. 2013). Our epistasis analysis, showing the nonadditive vic phenotype of Δatg1Δpmk1 double mutant and the resemblance of the Δatg1Δpmk1 double mutant to the Δpmk1 single mutant, suggests that Atg1 acts upstream of the Pmk1 signaling pathway. Furthermore, suppressioin of the toxicity induced by Pck2 overexpression by atg1 deletion suggests that Atg1 acts downstream of Pck2. MAPK signaling cascades consist of a core module of three tiers of protein kinases MAPK, MAPKK, and MAP3K, and often an additional upstream MAP4K. It would be intriguing if Atg1 serves as an additional layer of kinase mediating MAPK signaling activation. Future studies will elucidate the mechanism and the functional significance of the genetic interaction between autophagy and MAPK signaling revealed by our yeast genetic screen. Given the highly conserved nature of autophagy and MAPK signaling in the fate of cell death and proliferation, this study will provide valuable information to understand human diseases associated with aberrant regulation of MAPK signaling and autophagy.

Yeast strains, media, and molecular biology
Schizosaccharomyces pombe strains and plasmids used in this study are listed in the Reagents section. The complete medium YPD and the minimal medium EMM have been described previously (Toda et al. 1996). FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). Standard genetic and recombinant-DNA methods (Moreno et al. 1991) were used except where noted.

vic mutant screening
The growth of 89 viable kinase knockout strains generated by Bimbo et al. (Bimbó et al. 2005) was analyzed on the YPD plates with or without FK506 and 0.09 M MgCl 2 by spotting growth assay.

Spotting growth assay
Yeast cells were cultured in 20 ml of liquid media at 27˚C till mid-log phase and diluted to 0.6 OD 660 /ml, from which five 10fold serial dilutions were prepared and then spotted onto the indicated plates. Plates were incubated at 27˚C for 3 to 7 days.  (Ma et al. 2006)